Summary of Work: Human mitochondrial DNA evolves faster and is more prone to oxidative damage than is nuclear DNA. Mutations in mitochondrial DNA causes a wide range of mitochondrial dysfunctional diseases affecting some 50 million people in the US. These mutations may occur during replication by the DNA polymerase gamma. AZT and dideoxynucleotides produce mitochondrial toxicity by inhibition of the DNA polymerase gamma. How the mitochondrial DNA polymerase makes mutations and what structural properties set this polymerase apart from the nuclear DNA polymerases to give rise to its inhibition patterns are poorly understood. To address these questions we previously cloned the human DNA polymerase gamma cDNA. Using our overproduced human DNA polymerase gamma we have discovered that the polymerase can perform translesion synthesis past cisplatinin adducts. We purified the native human DNA polymerase complex and identified the smaller subunit to be 55 kDa. The cDNA for the Hp55 was isolated and overexpressed in E. coli and protein purified to homogeneity. The Hp55 accessory protein stimulates DNA polymerase activity, confers highly processive DNA synthesis to DNA polymerase gamma by providing greater affinity to DNA, binds DNA with tight affinity in the absence of catalytic polymerase subunit, and protects DNA polymerase gamma from N-ethylmaleimide inhibition. The fidelity of DNA synthesis of the wild type and exonuclease deficient DNA polymerase was determined and we find that the catalytic subunit has a large propensity to make errors in homopolymeric runs. We have isolated a new gene in yeast that regulates mitochondrial copper levels. Deletion of this gene results in slow growth, increased sensitivity to copper, and a dramatic increase in the rate of loss of mitochondrial function. This gene is highly conserved, and a human homolog has been identified. Mutation of the human gene could result in a phenotype similar to other diseases with defective mitochondrial copper regulation, e.g. Friedreichs ataxia or Wilsons disease. - Catalysis, Gene Library, Hominidae, Nucleotide Mapping, Point Mutation, Saccharomyces Cerevisiae